MS2 Reporter-Ion Quantitation With Ratio Distortion Correction
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Solution Overview
Problem
Existing multiplexed quantitation methods using isobaric mass tags in mass spectrometry suffer from ratio distortion due to co-fragmentation of target and interfering ions, leading to inaccurate peptide abundance measurements.
Innovation Solution
A computational technique decomposes the mass spectrometric signal into components correlated and uncorrelated with the peptide of interest, using non-negative least squares analysis to correct reporter-ion ratios and reduce ratio distortion in MS2 analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If MS2 analysis is used for multiplexed quantitation with isobaric mass tags, then high-throughput analysis is achieved, but ratio distortion occurs due to co-fragmentation of target and interfering ions leading to inaccurate measurements
Solution Approach 1:
The patent segments the mass spectrometric signal into multiple components using computational decomposition. The reporter ion signal is separated into portions contributed by different precursor ions (target peptide vs. interfering peptides) based on their distinct fragmentation patterns and relative abundances across multiple MS2 scans. This segmentation allows accurate quantitation by isolating the target peptide's contribution from the composite signal.
Solution Approach 2:
The patent employs an iterative feedback approach where the decomposed signal components are used to refine the estimation of target peptide abundance. The method uses the observed reporter ion intensities and precursor ion abundances from multiple scans to calculate and adjust the contribution of each component, progressively improving the accuracy of the quantitation through feedback loops in the computational analysis.
2Measurement precision
If MS3 analysis is used to eliminate ratio distortion, then measurement accuracy is improved, but analysis speed decreases significantly
Solution Approach 1:
The patent replaces the mechanical/physical MS3 isolation and fragmentation approach with a computational signal processing method. Instead of performing an additional physical separation step (MS3), the invention uses mathematical decomposition of the MS2 signals to isolate the target peptide's contribution, achieving the same accuracy improvement without the time penalty of slower MS3 analysis.
Data Source
AI summary
A method for correcting abundance ratios between pairs of isobaric reporter ions comprises: (a) measuring, for each liberated reporter-ion moiety the variation, with time, of a signal from said moiety; (b) identifying a first set and a second set of reporter-ion moieties for which the respective signal is, respectively, positively correlated with and not correlated with, the time variation of one or more other signals or variables that pertain to the detection of one or more peptides of interest; (c) for each reporter-ion moiety, decomposing the respective measured mass spectrometric signal into first and second portions that, respectively are and are not attributable to the peptide; (d) for each identified reporter-ion moiety, setting a respective adjusted mass spectrometric signal as being the respective portion of the signal that is attributable to the peptide; and (e) calculating corrected reporter-ion ratios based on the adjusted mass spectrometric signals.